Evolutionary rewiring of transcriptional networks is temporally associated with a putative 'Shield and Sword' pathogenicity strategy

Fungal specialists and generalists employ distinct pathogenic strategies. While Botrytis cinerea is a well-studied generalist, the mechanisms of the host-restricted specialist Botrytis deweyae remain largely unexplored. We integrated comparative genomics, temporal in planta transcriptomics, and targeted metabolomics to evaluate the virulence strategies of B. deweyae . Comparative genomic analysis of the host-restricted specialist Botrytis deweyae revealed structural genome streamlining, confirming chromosome-level genomic contractions and retaining a specialized set of 2,581 predicted protein-coding genes lacking high-identity orthologs in Botrytis cinerea . Re-analysis of host infection transcriptomes on Hemerocallis using PyDESeq2 identified a biphasic 'Shield and Sword' virulence model across three sequential time points (24, 48, and 96 hours post-infection, hpi). The early defensive 'Shield' phase (24 hpi; 783 down-regulated and 999 up-regulated genes, , ) is characterized by the upregulation of oxidative stress response pathways, including a species-specific 6-phosphogluconate dehydrogenase (EAE98_008955; , ), which corresponds to a significantly higher phenotypic tolerance to exogenous in B. deweyae compared to B. cinerea . At 48 hpi (1,630 down-regulated and 1,566 up-regulated genes), the fungus shifts to a metabolic offensive ('Sword' phase), upregulating secondary metabolism genes. This transition is temporally associated with a candidate zinc-finger transcription factor (EAE98_002040; , ) coordinating a co-regulated transition module of 18 genes. By 96 hpi (768 down-regulated and 813 up-regulated genes), a late necrotrophic module is activated, temporally associated with a late-stage zinc-finger transcription factor (EAE98_010455; , ) that co-express with 16 genes. Targeted metabolomics show B. deweyae overproduces botryane-type sesquiterpenes, with the Bcbot2 toxin biosynthesis gene strongly upregulated in the dark, contrasting with light-dependent production in B. cinerea . These results suggest that transcriptional rewiring coordinates a specialized host-infection strategy in B. deweyae .